Team:UCSF/Project/Background2

From 2013.igem.org

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{{Template:UCSF/MainHeader}}
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    <!----{{Template:12SJTU_scroll2top_css}}---->
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<html>
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<head>
<head>
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<!--CSS styles: global-->
<style type="text/css">
<style type="text/css">
-
.back-to {
+
/***
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    position: fixed;
+
Minimal header:  
-
    bottom: 35px;
+
Thanks a lot to 2012 Calgary team for snippets of their code!
-
    *bottom: 50px;
+
Check out their wikis at:
-
    right: 10px;
+
https://2012.igem.org/Team:Calgary
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+
***/
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</html>
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    <!----End {{Template:12SJTU_scroll2top_css}}---->
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    <!----{{Template:12SJTU_sliderbar}}---->
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#content h1.firstHeading {
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    <!----End {{Template:12SJTU_sliderbar}}---->
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visibility:hidden;
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    <!----{{Template:12SJTU_theme_css}}---->
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    <html>
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<head>
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<style type="text/css">
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table.head
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box-shadow: 0px 0px 16px rgba(0, 0, 0, 0.4);
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table.main
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#p-logo {
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{
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display: none;
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border-right: medium none;
+
}
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border-top: medium none;
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#searchform {
-
border-left: medium none;
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    display: none;
-
border-bottom: medium none;
+
}
}
-
table.mainouter
+
.left-menu {
-
{
+
background-color: #555;
-
width:1012px !important;
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position:relative;
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box-shadow: 0px 10px 16px rgba(0,0,0,0.4);
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}
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td.pumpkin
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-
{
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-
height: 100px;
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-
width: 1012px;
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background-color: black;
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}
}
-
#floatpumpkin
+
 
-
{
+
div#top-section{ /*the div containing the entire top bar*/
-
position:absolute;
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height: 20px;
-
left:74px;
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margin-bottom: 0px !important;
-
top:0px;
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border: none;
-
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-
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-
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}
}
-
/*-- Lavalamp --*/
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-
.navipages:after { visibility: hidden; display: block;}
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-
*:first-child+html .navipages { zoom: 1; } /* IE7 */
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#content{
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#lavalampbg {
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margin-top: 0px;
-
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background-image:url(/wiki/images/4/4d/12SJTU_pumpkin.png);
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z-index: 100;
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}
}
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table.message
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-
{
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#search-controls {
-
border:transparent 1px solid;
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-
}
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table.bottom
+
background: none;
-
{
+
position: absolute;
-
background: none transparent scroll repeat 0% 0%
+
top: 170px;
 +
right: 40px;
 +
}  
 +
 
 +
 
 +
div#header {
 +
width: 975px;
 +
text-align: left;
 +
margin-left: auto;
 +
margin-right: auto;
 +
margin-bottom: 0px !important;
 +
} 
 +
 
 +
#menubar {
 +
position: absolute;
 +
background: none;
 +
color: black;
}
}
-
p
+
 
-
{
+
.left-menu, .right-menu{
-
font-size:106%;
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-
text-align:justify;
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dd
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a.info
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border-bottom: #0cf 1px solid;
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position: absolute;
position: absolute;
-
top: 2em;
+
background: none;
-
background-color: #cff;
+
color: black;
-
text-align: center
+
}
}
-
table.bottom
+
 
-
{
+
.left-menu li a, .right-menu li a {
-
background: none transparent scroll repeat 0% 0%
+
color: #000 !important;
}
}
-
h1 + p {
+
 
-
padding-bottom: 1em;
+
 
 +
.left-menu ul li, .right-menu ul li a{
 +
background: none;
 +
color: #000 !important;
}
}
-
h1 + p a b {
+
 
-
font-weight:normal!important;
+
.left-menu li a:hover, .right-menu li a:hover, .right-menu li a:visited, .right-menu li a:active {
 +
    color: #000 !important;
}
}
-
td
+
#catlinks{
-
{
+
display:none;
-
border:1px solid transparent;
+
}
}
-
td.embedded
+
/*important for background colours*/
-
{
+
.mediawiki{
-
border-right: medium none;
+
background: #ffffff;
-
padding-right: 0px;
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-
border-top: medium none;
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+
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border-bottom: medium none;
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text-align: left
+
}
}
-
td.bottom
+
 
-
{
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/***End minimal header***/
-
border-right: medium none;
+
 
-
border-top: medium none;
+
/*Base styles*/
-
border-left: medium none;
+
#content{
-
border-bottom: medium none
+
border: none;
}
}
-
td.heading
+
h1, h2,h3, h4, #css-full, #css-mobi{
-
{
+
font-family: Myriad Pro, Gill Sans MT, Trebuchet MS, Arial, Sans-Serif;
-
font-weight: bold
+
border: 0;
 +
font-weight: normal;
}
}
-
td.outer {
 
-
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td.text
 
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{
 
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-
padding-top: 10pt;
 
-
background: white;
 
-
}
 
-
td.text[align=center] {
 
-
padding-right: 0 !important;
+
p1, p2, p3, div.thumb div div.thumbcaption{
-
padding-left: 0 !important;
+
font-family: Calibri, Sans-Serif;
-
padding-bottom: 0 !important;
+
font-weight: normal;
-
/*padding-top: 5px!important;*/
+
color: black;
-
padding-top:0 !important;
+
-
background-color:black;
+
}
}
-
td.colhead,h2{
+
#css-full, #css-mobi{
-
color: #ffffff;
+
position: absolute;
-
border-bottom: 1px solid #AAA;
+
float: right;
-
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+
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-
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+
font-size: 1.3em;
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background-color: #ECECEC;
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top: 0px;
-
font-size:13pt;
+
right: 15px;
-
color:black;
+
display: block;
 +
padding: 10px;
}
}
-
h3
+
#jsnotice{
-
{
+
background-color: #4ED92F;
-
font-size:11pt;
+
-
padding-top: 0px;
+
-
padding-bottom: 0px;
+
}
}
-
h4
 
-
{
 
-
font-size: 5pt;
 
-
font-family: Arial;
 
-
font-weight: bold;
 
-
}
 
-
td.rowhead_left
 
-
{
 
-
text-align: left
 
-
}
 
-
td.rowhead_center
 
-
{
 
-
text-align: center
 
-
}
 
-
td.rowhead
 
-
{
 
-
font-weight: bold;
 
-
vertical-align: top;
 
-
text-align: right
 
-
}
 
-
t
 
-
td.navigation
+
#table{
-
{
+
margin: 10px;
-
border-right: medium none;
+
-
border-top: medium none;
+
-
font-weight: bold;
+
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+
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+
-
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td.clear
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td.nothing
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+
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font.gray
+
a:link,
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{
+
a:visited{
-
color: #aca899;
+
font-family: Calibri, Sans-Serif;
-
text-decoration: underline
+
font-weight: normal;
 +
color: #4E9600;
 +
text-decoration:none;
}
}
-
a:link
 
-
{
 
-
text-decoration: none
+
a:hover,
 +
a:active{
 +
font-family: Calibri, Sans-Serif;
 +
font-weight: normal;
 +
color: #4E9600;
 +
text-decoration:underline;
}
}
-
li a{
+
-
color: #000000;
+
/***Body styling***/
 +
h1{
 +
font-size: 2.2em;
 +
line-height: 1.2em;
 +
color: #008000;
 +
font-weight: bold;
}
}
-
td.colhead a {
+
h2{
-
color: #000000;
+
font-size: 2.0em;
 +
line-height: 1em;
 +
color: #008000;
 +
font-weight: bold;
}
}
-
td.colhead a:hover {
+
h3{
-
color: #FFFFFF;
+
font-size: 1.8em;
 +
line-height: 1em;
 +
//margin: 0px 15px;
 +
//font-weight: bold;
 +
        text-decoration: underline;
 +
color: #008000;
}
}
-
a:visited
+
h4{
-
{
+
font-size: 1.6em;
-
text-decoration: none
+
color: #333333;
 +
margin: 0px 20px;
 +
font-weight: bold;
}
}
-
a:hover
+
p1{
-
{
+
        font-size: 1.7em;
-
/* TODO */
+
color: #008000;
-
color: #777;
+
font-weight: bold;
}
}
-
a.index
+
p2{
-
{
+
        font-size: 1.5em;
-
font-weight: bold
+
}
}
-
 
+
p3{
-
img.border
+
        font-size: 1.2em;
-
{
+
-
border-right: #000000 1px solid;
+
-
border-top: #000000 1px solid;
+
-
border-left: #000000 1px solid;
+
-
border-bottom: #000000 1px solid
+
}
}
-
.mainouter tbody tr:first-child td.text .shadetabs ul {
+
#pagetitle{
-
text-align: left!important;
+
border-bottom: 2px solid black;
 +
padding-bottom: 10px;
 +
padding-left: 10px;
}
}
-
 
+
#bodycontainer h2{
-
 
+
margin-left: 10px;
-
</style>
+
margin-right: 10px;
-
</head>
+
-
</html>
+
-
    <!----End {{Template:12SJTU_theme_css}}---->
+
-
 
+
-
<html>
+
-
<head>
+
-
<style type="text/css">
+
-
 
+
-
 
+
-
#p-logo{
+
-
  display: none;
+
}
}
-
#footer-box{
+
#bodycontainer p{
-
  display: none;
+
margin-left: 20px;
 +
margin-right: 10px;
}
}
-
div.thumb {
+
#bodycontainer{
-
    margin-bottom: .5em;
+
margin-left: 220px;
-
    border-style: solid;
+
-
    border-color: transparent;
+
-
    width: auto;
+
}
}
-
#menubar li{
+
#bodycontainer ul{
-
    margin-left: 0px;
+
margin-left: 5.0em;
}
}
-
#firstHeading{
+
#bodycontainer li{
-
    display: none;
+
font-family: Georgia, Serif;
}
}
-
#top-section{
+
#box1{
-
    border: 0px;
+
width: 740px;
-
}
+
        height: 2000px;
-
#content{
+
background: #fffff;
-
    background: none;
+
float: left;
-
    border: 0px;
+
        margin-left: 210px;
-
}
+
        margin-top: 15px;
-
#contentSub{
+
-
    display: none;
+
-
}
+
-
.firstHeading{
+
-
    display: none;
+
-
}
+
-
#catlinks{
+
-
    display: none;
+
}
}
-
#globalWrapper{
 
-
margin: 0px 0px 0px 0px;
 
-
height: 0px;
 
-
background-repeat:no-repeat !important;
 
-
background-attachment: fixed!important;
 
-
color: #000000;
 
-
background-color: #e6e6e6;
 
-
-moz-background-size:cover !important;
 
-
background-size:cover !important;
 
-
padding:0px 0px 0px 0px;
 
-
}
 
-
#main_wrapper{
 
-
position:absolute;
 
-
top:-93px;
 
-
left:-20px;
 
-
margin: 0px 0px 0px 0px;
 
-
width:1012px;
 
-
background-repeat:no-repeat !important;
 
-
background-attachment: fixed!important;
 
-
color: #000000;
 
-
-moz-background-size:cover !important;
 
-
background-size:cover !important;
 
-
padding-top:8px;
 
-
}
 
-
#search-controls
 
-
{
 
-
display:none;
 
-
}
 
</style>
</style>
-
    <script src="http://igem.bio-x.cn/home/style/jquery-1.3.2.min.js" type="text/javascript"></script>
 
-
    <script type="text/javascript" src="http://ajax.googleapis.com/ajax/libs/jquery/1.4/jquery.min.js?ver=1.3.2"></script>
 
-
    <script type="text/javascript">
 
-
        jQuery.noConflict();
 
-
        ($(function () {
 
-
            var $el, leftPos, newWidth,
 
-
$mainNav = $("#lavalamp");
 
-
            $mainNav.append("<li id='lavalampbg'></li>");
 
-
            var $magicLine = $("#lavalampbg");
 
-
            $magicLine
 
-
.css("left", $(".current a").position().left + $(".current a").width() / 2 - 35)
 
-
.data("origLeft", $(".current a").position().left + $(".current a").width() / 2 - 35)
 
-
            $("#lavalamp li").find("a").hover(function () {
 
-
                $el = $(this);
 
-
                leftPos = $el.position().left + $el.width() / 2 - 35;
 
-
                $magicLine.stop().animate({
 
-
                    left: leftPos
 
-
                })
 
-
            }, function () {
 
-
                $magicLine.stop().animate({
 
-
                    left: $magicLine.data("origLeft")
 
-
                });
 
-
            });
 
-
        })
 
-
); (jQuery);
 
-
</script>
 
-
</head>
+
<!------------------------------Context Styles-------------------------------->
-
<body>
+
<style>
 +
#mission {width: 500px; float:left; background-color: #F5F5F5; margin-left:8px; padding: 10px; margin-top:8px;}
 +
#opensource {width:306px; float:left; background-color: #F5F5F5; margin-left:8px; padding: 10px; margin-top:8px;}
 +
#rightcontent {width:800px; float:right; background-color: #F5F5F5; margin-left: 8px;  margin-top:10px;}
 +
#photos {float:left; background-color: #FFFFFF; margin-left: 15px;  margin-top:5px;}
 +
#leftcontenttext{float:left; background-color: #FFFFFF; margin-left: 15px;  margin-top:5px;}
 +
#rightcontenttext {float:right; background-color: #FFFFFF; margin-left:15px; padding:10px; margin-top:0px;}
 +
//#flickr{width:755px; float:right;} 
 +
</style>
-
<div id="main_wrapper">
 
-
<table class="head" cellspacing="0" width="1012" cellpadding="0" align="center"><tbody>
 
-
<td><tr></tr>
 
-
</td>
 
-
</tbody></table>
 
-
 
-
+
<!---------------------------------Sidebar------------------------------------------>
-
<!--       =============================================================================== -->
+
<!--Thanks a lot to 2012 SJTU-BioX-Shanghai team for snippets of their code!
 +
Check out their wikis at:
 +
https://2012.igem.org/Team:SJTU-BioX-Shanghai
 +
-->
-
 
+
<!--Sidebar Scripts-->
-
<div id="toolBackTo" class="back-to" style="display:block;">
+
-
<a class="backtotop" href="#top" onclick="window.scrollTo(0,0);return false;">To Top
+
-
+
-
</a>
+
-
</div>
+
-
 
+
-
</body>
+
-
</html>
+
-
<!--End {{Template:12SJTU_header}}-->
+
-
 
+
-
<!--{{Template:12SJTU_nav_project}}-->
+
-
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         <span>Overview</span>
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__NOTOC__
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=Project Overview=
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Advance in molecular cloning technology has made it possible for mankind to entitle engineered organisms to different biochemical reactions. However, the speed of those enzymatic reactions is often limited because intermediates produced from upstream enzyme cannot be passed efficiently to downstream enzyme due to spatial obstacles. Thus, synthetic scaffold built to decrease distance between enzymes for speeding biochemical reactions is a rising topic with promising application prospect.
+
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Moreover, although some progress has been made in fields of metabolic flux control with synthetic scaffold, these strategies remain  non-dynamic. Artificially and dynamically controlling metabolic flux has remained a challenge.
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<h1><center> Operation CRISPR: Deploying precision guided tools to target unique species in a complex microbiome </center></h1>
 +
 
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 +
<p2>Rarely in nature do bacterial strains exist in isolation; they form complex microbial communities that interact with various organisms. We ourselves contain a major microbial community in our digestive tract that has shown to directly affect our health and well-being.  As shown on the left, to improve and maintain healthly living it would be useful to have the ability to change the microbial community. For example, if a large of amount of a certain sugar was present in your gut ("signal #1") you might want to slow the growth of a certain bacterial populations . In another scenario ("signal #2") it might be useful to increase the growth of other specific bacteria in your gut. But targeting precise bacterial community strains and controlling their growth, activity, and outputs is difficult and requires many new tools.</p2>
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==Introduction==
+
<div id="photos">
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<center><img style="height:290px; margin-left:150px" src="https://static.igem.org/mediawiki/2013/1/15/IntroMicrobiome_Pic1.png"> </center>
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In this year, we expanded the definition of ''scaffold'' in synthetic biology and developed two universal devices called ''Membrane Accelerator'' and  ''Membrane Rudder'' respectively. Together, they made ''Membrane Magic'' happen!
+
<div id="leftcontenttext" style = "width:740px; height:280px">
 +
<p2>At the beginning of this summer, we asked ourselves a question: "What could we introduce to a microbiome which would allow targeting and eventual gene expression changes in a specific bacteria?" The difficulty faced with this situation is in:</p2>
 +
<p2>  1. Introduce a targeting system into a defined mixture of bacteria such that you can select and introduce manipulations without negatively affecting other bacteria. </p2>
 +
<p2> 2. Creating easy to transfer pathways or circuits that can produce a multitude of outcomes (killing, repressing, upregulating). </p2>
 +
<p2><br><br></p2>
 +
<h3>1. Introducing CRISPRi to a bacterial community</h3>
 +
<p2>To selectively target and eliminate harmful bacteria, we are utilizing the CRISPRi system, a tool repurposed from a natural adaptive immunity system in bacteria (see diagram below). This tool is comprised of a catalytically dead Cas9 (dCas9) protein that complexes with guide RNAs (gRNA) complementary to the target bacteria’s DNA sequence. This complex binds to DNA complementary to the gRNA and prevents transcription, therefore repressing gene expression.</p2>
 +
</div>
-
Previous researchers have focused on building protein, RNA or DNA scaffold as constitutive assemblies carrying enzymes. They have  succeeded in increasing product yields. However, the amount of those scaffolds could be limited by its expression or copy level, leading to restriction on further acceleration. With ''Membrane Magic'', we made ''E.coli'' membrane into a huge scaffold accommodating enzymes without limitation of scaffold amount. Moreover, protein assembly on membrane could readily receive extracellular or intracellular signal, so the whole system becomes highly tunable. The superiority of Membrane Scaffold is shown in details in '''WHY MEMBRANE'' section below.
+
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 +
<p2>WHY USE CRISPRi? <br><br>1. CRISPRi utilizes gRNAs which are highly specific and customizable.<br><br>
 +
2. In principle it could be used to take advantage of unique DNA sequences to target specific bacterial species.</p2>
 +
</div>
-
One of our devices, called ''Membrane Accelerator'', functions by localizing and organizing enzymes on membrane surface. ''E.coli'' inner membrane serves as a two-dimensional plane that can accommodate various protein assemblies linked with enzymes. Otherwise diffusing enzymes can form clusters on membrane through interacting protein domains and ligands. Enzyme clusters help substrates flow between enzymes, and  thus increase yields of sequential biological reactions. We not only applied the ''Membrane Accelerator'' into biosynthetic  pathway but also biodegradation pathway, which is proposed for the first time in synthetic biology. Previous researches on synthetic scaffold controlling metabolic flux all focused on biosynthesis.
+
<div id="photos" style = "width:340px">
 +
<img style="height:400px; margin-left:40px; padding:0;"src="https://static.igem.org/mediawiki/2013/a/a6/CRISPRi_Background_UCSF-1.png">
 +
</div>
 +
<div id="leftcontenttext" style = "width:300px; height:20px; margin-left:55px; margin-top:-5px" align="justify">
 +
<p3><center>Amended from: http://www.cell.com/abstract/S0092-8674(13)00211-0?script=true</center></p3>
 +
</div>
-
[[File:12SJTU membrane accelerator sketch.jpg|thumb|400px|center|''Fig.1:'' Sketch of ''Membrane Accelerator'']]
+
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 +
<p2> As a means <a href="https://2013.igem.org/Team:UCSF/Project/Conjugation/Design1" > to introduce our CRISPRi system into a microbial community we've opted to utilize  conjugation </a> - a naturally occurring mechanism bacteria use to transfer DNA.  By utilizing this mechanism, we are able to target specific strains of bacteria and affect gene expression. This will have a potential for future applications that require targeting individual strains in a bacterial community. </p2></div>
-
Although some work has been done in reaction acceleration, it has always been a challenge to artificially and dynamically control those reactions. Our ''Membrane Rudder'' device, however, offers a novel method to control the direction of biochemical reactions through varieties of signals. We further combined the whole post-translational control system with genetic circuits by recruiting RNA aptamer and its corresponding binding protein. Thus RNA signal could also be recruited to dynamically control biochemical reaction.
+
<div id="photos" style = "width:340px"; height:155px;>
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[[File:12SJTU_overview4.gif|center|400px]]
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 +
src="https://static.igem.org/mediawiki/2013/8/8b/Conjugation_Bkgrd_UCSF-1.png"> </center><br></div>
-
<center>''Fig.2:'' Sketch of ''Membrane Rudder''</center>
+
<div id="leftcontenttext" style = "width:740px; height:80px; margin-top:10px"align="justify">
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<br\>
+
<p1><center>The combination of conjugation and CRISPRi allows us to create a system capable of both transferring genetic instructions from one cell to another as well as targeting unique species in a microbial community through a specific gene. </center></p1>
-
<br\>
+
</div>
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<br\>
+
-
==Why MEMBRANE?==
+
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-
 
+
<h3>2. Creating scalable CRISPRi circuits that can choose between outcomes based on the input</h3>
-
Why do we choose membrane as our primary scaffold to assemble enzymes?
+
<p2>In addition to our conjugation project, we have developed a <a href="https://2013.igem.org/Team:UCSF/Project/Circuit/Design1" >CRISPRi circuit</a>, which could be delivered by the same conjugation system, that could apply to future regulatory applications (upregulation of bacterial growth, bacterial activity and behavior, gene expression, and other bacterial processes, etc.). Our circuit is multi-functional, eliciting different responses with the presence of different inducers and is scalable by incorporating additional designed plasmids or guide RNAs. The circuit relies on the use of CRISPRi gRNAs to provide scalability - several genes can be targeted for silencing, upregulation, or other needs. </p2>
-
{|
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|-
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|1. '''Natural Scaffold: ''' Different from previous synthesized scaffold, membrane scaffold is an innate one. Besides, there is no limitation on scaffold amount.
+
</body>
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|[[File:12SJTU Why membrane1.png|300px|right|thumb|''Fig.3:'' Natural Scaffold]]
+
-
|-
+
-
|2. '''Two-Dimensional Plane: ''' Membrane Scaffold changes restricted the reaction space to a two-dimensional plane compared to discrete scaffold. Thus proteins on membrane are more likely to interact with each other (Demonstrated in [https://2012.igem.org/Team:SJTU-BioX-Shanghai/Project/project2.2 Fatty Acid Synthesis:The Refinement of Interaction]). Moreover, we can organize enzymes in 2D pattern on membrane to further facilitate metabolic flux (Demonstrated in [https://2012.igem.org/Team:SJTU-BioX-Shanghai/Project/project2.3 DBT desulfurization]).
+
-
|[[File:12SJTU Why membrane2.jpg|300px|right|thumb|''Fig.4:'' Two-Dimensional Plane]]
+
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|-
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|3. '''Priority to Exportation: ''' Concentration of final products could be effectively increased near the membrane with Membrane Scaffold, which in turn, facilitates the transmembrane transportation. Thus final products would be more readily to be exported to extracellular media. (Demonstrated in [https://2012.igem.org/Team:SJTU-BioX-Shanghai/Project/project2.2 Fatty Acid Synthesis:The Priority to Exportation])
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|[[File:12SJTU Why membrane3.jpg|300px|right|thumb|''Fig.5:'' Priority to Exportation]]
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|4. '''Ability to Sense Signals: ''' Membrane Scaffold provides a platform to directly receive environmental and internal signal, So biochemical reactions could be dynamically controlled through those signals(Demonstrated in [https://2012.igem.org/Team:SJTU-BioX-Shanghai/Project/project1.3 Membrane Rudder Design] and [https://2012.igem.org/Team:SJTU-BioX-Shanghai/Project/project2.1 Violacein pathway: Membrane Rudder Application]).
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|[[File:12SJTU Why membrane4.jpg|300px|right|thumb|''Fig.6:'' Ability to sense signals]]
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==Reference==
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1.Delebecque, C. J., A. B. Lindner, et al. (2011). "Organization of intracellular reactions with rationally designed RNA assemblies." Science 333(6041): 470.
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2.Dueber, J. E., G. C. Wu, et al. (2009). "Synthetic protein scaffolds provide modular control over metabolic flux." Nature biotechnology 27(8): 753-759.
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3.Conrado, R. J., G. C. Wu, et al. (2012). "DNA-guided assembly of biosynthetic pathways promotes improved catalytic efficiency." Nucleic acids research 40(4): 1879-1889.
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Latest revision as of 03:56, 29 October 2013

Operation CRISPR: Deploying precision guided tools to target unique species in a complex microbiome

Rarely in nature do bacterial strains exist in isolation; they form complex microbial communities that interact with various organisms. We ourselves contain a major microbial community in our digestive tract that has shown to directly affect our health and well-being. As shown on the left, to improve and maintain healthly living it would be useful to have the ability to change the microbial community. For example, if a large of amount of a certain sugar was present in your gut ("signal #1") you might want to slow the growth of a certain bacterial populations . In another scenario ("signal #2") it might be useful to increase the growth of other specific bacteria in your gut. But targeting precise bacterial community strains and controlling their growth, activity, and outputs is difficult and requires many new tools.
At the beginning of this summer, we asked ourselves a question: "What could we introduce to a microbiome which would allow targeting and eventual gene expression changes in a specific bacteria?" The difficulty faced with this situation is in: 1. Introduce a targeting system into a defined mixture of bacteria such that you can select and introduce manipulations without negatively affecting other bacteria. 2. Creating easy to transfer pathways or circuits that can produce a multitude of outcomes (killing, repressing, upregulating).

1. Introducing CRISPRi to a bacterial community

To selectively target and eliminate harmful bacteria, we are utilizing the CRISPRi system, a tool repurposed from a natural adaptive immunity system in bacteria (see diagram below). This tool is comprised of a catalytically dead Cas9 (dCas9) protein that complexes with guide RNAs (gRNA) complementary to the target bacteria’s DNA sequence. This complex binds to DNA complementary to the gRNA and prevents transcription, therefore repressing gene expression.
WHY USE CRISPRi?

1. CRISPRi utilizes gRNAs which are highly specific and customizable.

2. In principle it could be used to take advantage of unique DNA sequences to target specific bacterial species.
Amended from: http://www.cell.com/abstract/S0092-8674(13)00211-0?script=true
As a means to introduce our CRISPRi system into a microbial community we've opted to utilize conjugation - a naturally occurring mechanism bacteria use to transfer DNA. By utilizing this mechanism, we are able to target specific strains of bacteria and affect gene expression. This will have a potential for future applications that require targeting individual strains in a bacterial community.

The combination of conjugation and CRISPRi allows us to create a system capable of both transferring genetic instructions from one cell to another as well as targeting unique species in a microbial community through a specific gene.

2. Creating scalable CRISPRi circuits that can choose between outcomes based on the input

In addition to our conjugation project, we have developed a CRISPRi circuit, which could be delivered by the same conjugation system, that could apply to future regulatory applications (upregulation of bacterial growth, bacterial activity and behavior, gene expression, and other bacterial processes, etc.). Our circuit is multi-functional, eliciting different responses with the presence of different inducers and is scalable by incorporating additional designed plasmids or guide RNAs. The circuit relies on the use of CRISPRi gRNAs to provide scalability - several genes can be targeted for silencing, upregulation, or other needs.

Retrieved from "http://2013.igem.org/Team:UCSF/Project/Background2"